Anti-inflammatory Molecular Mechanism Of20（s）-ginsenoside Rh₂: Sulfated Derivative In Vitro

Ginsenosides is extracted from Ginseng and regarded as the the main activeingredient. It has been reported that ginsenosides have various pharmacologicalproperties such as anti-inflammatory, anti-tumor and antioxidant. In order to obtainginsenoside-like derivatives with lower toxicity and stronger biologic activity, wemodified ginsenoside Rh₂and got two new derivatives, which were named asderivative B₁（Rh₂-B₁）and derivative B₂（Rh₂-B₂）. They were proved to have betterwater-solubility. We built inflammatory model by LPS-induced murine RAW264.7macrophages in vitro so as to investigate the anti-inflammatory activity of ginsenosideRh₂derivatives, and further explored the anti-inflammatory molecular mechanism ofginsenoside Rh₂derivatives by inflammatory signal transduction pathway.Lipopolysaccharide （LPS） can induce macrophages to produce a number ofcytokines and other inflammatory mediators, which leading to several inflammatorydiseases. We first investigated the effect of different concentrations of ginsenoside Rh₂derivatives on cytokines and inflammatory mediator secretions in LPS-inducedmurine RAW264.7macrophages in vitro, and we found that ginsenoside Rh₂derivatives inhibited production of TNF-α, IL-6and IL-1β and increased IL-10secretion level in a dose-dependent manner （P<0.05or P<0.01）, which consistent withtheir mRNA expression. In addition, ginsenoside Rh₂derivatives also inhibitedsynthesis of NO and PGE₂, and reduced levels of iNOS and COX-2mRNA andprotein expression in RAW264.7macrophages in a concentration-dependent manner（P<0.05or P<0.01）. These results suggest that ginsenoside Rh₂derivatives may exertanti-inflammatory effect by regulating the synthesis and secretion of cytokines andinflammatory mediators in the inflammatory process.MAPKs and NF-κB are the most classical signal transduction pathway inLPS-induced inflammatory response. MAPKs consists of three main members thatinclude p38, JNK, ERK1/2, they were involved in LPS-mediated intracellular signaltransduction and cytobiological response. The other extracellular signal transductionpathway is the NF-κB pathway. NF-κB is one of the most ubiquitous transcription factors and participate in immune response, inflammatory response， cellularproliferation and apoptosis. In response to extracellular stimuli such as LPS, TNF-αor other inflammatory mediators, the transcription factor NF-κB is then translocatedinto the nucleus, where it attaches to κB binding sites in the promoter regions of targetgenes. This binding induces transcription of pro-inflammatory mediators andinflammatory response. In order to study anti-infalmmatory molecular mechanism ofginsenoside Rh₂derivatives, we further investigated the effect of ginsenoside Rh₂derivatives on MAPKs and NF-κB signal transduction pathways in LPS-inducedmurine RAW264.7macrophages. The results showed that as compared with LPScontrol group, ginsenoside Rh₂derivatives markedly inhibited LPS-induced p38andJNK protein phosphorylation in a dose-dependent manner, Rh₂-B₁also inhibitedERK1/2protein phosphorylation （P<0.05or P<0.01）. Furthermore, ginsenoside Rh₂derivatives obviously inhibited LPS-induced phosphorylation of IκBα and p65, on thecontrary, significantly increased IκBα and p65compared to LPS group （P<0.01）.These results suggested that ginsenoside Rh₂derivatives inhibited cytokine TNF-α,IL-6, IL-1β, NO and PGE₂secretion by regulating both NF-κB and p38, JNK,ERK1/2MAPKs pathways.This study is the first to show that ginsenoside Rh₂derivatives hasanti-inflammatory effect in vitro through regulating inflammation mediated by NF-κBand p38, JNK, ERK1/2MAPKs signal pathways, it will provide theoretical basis forreasonable application of ginsenoside Rh₂derivatives in clinic and improve its clinicalvalue.